Recombinant DNA Techniques chapter 18

1. Recombinant DNA Techniqueschapter 18 Part I

2. techniques and their applications. • Restriction Digest (to be done in lab) • Southern Blot • Northern Blot • Western Blot • Cloning

3. 1. Restriction Digest: The Cutting of DNA Endonucleases = Restricition Enzymes

6. EXAMPLES OF DNA AGAROSE GELS Ladder, marker or standard of known DNA sizes

7. An Application of Restriction DigestChromosomal Disorders

8. Put your thinking cap on!

9. Wild type Hb Sickle cell Hb Restriction enzyme 1 band is too small to view here) 2 3 Restriction enzyme loss of cutting site

10. Wt= wildtype M= mutated M M Wt Wt Wt M

11. An Application to Forensic & Paternity Studies 2. The Southern Blot

12. 1. Restriction Digest & gel electrophoresis TRANSFER DNA crime scene DNA suspect 2 DNA suspect 1 DNA victim

13. You must understand the reason for the “probe”!

14. But there are thousands of fragments!

15. So sometimes you have to really search for the differences between DNA.

16. Probe is complementary “hot” DNA

17. From: Restriction digest To result SUMMARY OF A SOUTHERN BLOT

18. Suspect 1 Suspect 2 Crime scene Victim Back to our crime scene

19. Techniques also are available to measure RNA and Protein 3. Northern Blot for RNA 4. Western Blot for Protein

20. Western Summary: Blotting techniques with probes Southern Northern

21. 5. Technique: Cloning of specific genes or DNA fragments = restriction enzyme + = ligase

22. Cut vector Cut foreign DNA or gene Paste together

23. 2 different antibiotic resistant genes Bacteria are now resistant to 2 antibiotics

24. Let’s apply these concepts to plasmid transformation.

25. Cut DNA here & insert foreign DNA

26. Will your transformed colonies with the insert be white or blue?

27. Summary of cloning DNA into a blue/white selection vector

28. End of part 1